As enzymes derived from microorganisms which hydrolyze keratan sulfate (hereinafter referred to as a keratan sulfate hydrolase), an endo-.beta.-galactosidase type enzyme which hydrolyzes galactosidic linkages in the sugar chain of keratan sulfate and an endo-.beta.-N-acetylglucosaminidase type enzyme which hydrolyzes N-acetylglucosaminidic linkages of that are known.
As microorganisms producing the endo-.beta.-N-acetylglucosaminidase type enzyme, only Ks 36 strain belonging to the genus Bacillus, which has been isolated from soil samples by the present inventors, is known, and the microbiological characteristics and enzymological properties thereof have been clarified (Japanese Patent Application Laid-open No. 2-57182).
Keratan sulfate is grouped into keratan sulfate I which exists in corneas of animals, and keratan sulfate II and keratan polysulfate which are contained in the tissues such as cartilages, and each of them is a copolymer comprising the disaccharide of galactose and N-acetylglucosamine as a constitutional unit. In the former, most of the 6-positions of N-acetylglucosamine have a hydroxyl group, while a large proportion of the constituents are the disaccharide disulfate of which both hydroxyl groups of the 6-positions of N-acetylglucosamine and galactose are sulfated in the latter.
A keratan sulfate hydrolase of endo-.beta.-N-acetylglucosaminidase type acts on these keratan sulfates above and mainly produces sulfated keratan a sulfate disaccharide and sulfated keratan sulfate tetrasaccharide as the hydrolyzates. The disaccharide includes monosulfated keratan sulfate disaccharide and disulfated keratan sulfate disaccharide whose reducing end sugars are N-acetylglucosamine.
Accordingly, the hydrolyzates of keratan sulfate by the keratan sulfate hydrolase of endo-.beta.-N-acetylglucosaminidase type are mainly sulfated keratan sulfate -disaccharide and sulfated keratan sulfate tetrasaccharide having the N-acetyllactosamine structure.
N-acetyllactosamine is contained in the sugar chain of oligosaccharide in human milk, lipopolysaccharides, and a variety of glycoproteins and glycolipid, known as a growth factor of Lactobacillus bifidus (Bifidus factor) which is an enterobacterium of breast-fed babies, and promisingly expected to be used for high nutritious foods such as prepared dried milk for babies. And recently, the oligosaccharide represented by the so-called sialyl Le.sup.x sugar chain which is produced by bonding fucose to the N-acetylglucosamine residue of N-acetyllactosamine by the .alpha.-1,3 bond and sialic acid to the galactose residue by the .alpha.-2,3 bond, has an inhibitory activity to cell adhesion and is expected to be a constituent of anti-inflammatory agents, and therefore N-acetyllactosamine is promisingly expected also as a starting material of the sialyl Le.sup.x sugar chain synthesis.
It is known that N-acetyllactosamine is prepared by a method of synthesis with an enzyme, a method of desulfation of sulfated keratan disaccharide which is a hydrolyzate with a keratan sulfate hydrolase, and so on. And it is known that sulfated N-acetyllactosamine tetrasaccharide is prepared by hydrolyzing keratan sulfate with a keratan sulfate hydrolase as described above. However, as the keratan sulfate hydrolase of endo-.beta.-N-acetylglucosaminidase type used in the preparation above, only the keratan sulfate hydrolase (keratan sulfate hydrolase II) derived from the Ks 36 strain is known as described above. This enzyme does not have enough thermostability as it is not stable at approximately 30.degree. C. or above; therefore, it is not considered an appropriate enzyme in order to prepare sulfated keratan sulfate disaccharide or tetrasaccharide on a large industrial scale. Especially in the preparation of sulfated keratan sulfate disaccharide or tetrasaccharide by a batch degradation method or immobilized enzyme degradation methods, the keratan sulfate hydrolase is required to have high thermostability, and the development of the keratan sulfate hydrolase of endo-.beta.-N-acetylglucosaminidase type having high thermostability has been desired.